When studying growth and death of a micro-organism under the influence of specific environments, e.g. production and storage environments that e.g. could or could not be refrigerated, or involving chemicals or matrixes, e.g. antibiotics, microbial toxins, heavy metals and serum complement, microbial cultures are most often incubated for hours or days. In these circumstances death and growth occur simultaneously. If additionally some of the cells lyse, e.g. when analysing the serum complement, it is difficult to know to what one should compare the amount of living cells at the, end of the experiment. Convenient methods to determine the number of living cells, e.g. by measuring luciferase bioluminescence, are known but if no more information is available it is impossible to assess to what extent growth or/and death of the micro-organisms takes or has taken place.
Growth rates and death rates of micro-organisms in specific environments are of interest in many areas. Death rates and growth rates of micro-organisms and especially harmful and/or pathogenic micro-organisms are of importance in risk assessments of products of the pharmaceutical industry and products for human consumption with regard to their production, storage and distribution to the consumers. Knowledge of death and growth rates of micro-organisms are of particular importance in specific applications such as in the development of antibiotics, disinfectants and bactericidal products or monitoring of sterilisation, disinfection and cleaning processes.
Reporter genes coding for luminescent or/and fluorescent products have been introduced to micro-organisms to enable the assessment of the quantity or survival of living micro-organisms (WO 96/23898, WO 98/14605, WO 98/30715, WO 98/36081, U.S. Pat. No. 5,824,468). Even simultaneous use of luminescent and fluorescent markers has been used (Fratamico et al., Journal of Food Protection, Vol 50 No 10, 1997, 1167-1173). Luminescent and fluorescent markers have, however, only been used as markers for survival of micro-organisms and the use of two different markers within one micro-organism enabling the differentiation between growth and death rates has not been reported.